Cellular microparticles are membrane microvesicles released in biological fluids during cell activation or during apoptosis, in the course of various pathological conditions, including inflammation or diseases associated with impaired vascular function.
These microparticles are released into the extracellular space after a remodelling of the cell membrane, thus exposing on the outer leaflet phosphatidylserine, which is normally present on the inner leaflet, as well as other identity markers of their cellular origin.
Cellular microparticles are submicron sized (0.1-1 μm). Although they are present in the biological body fluids, especially in the circulating blood of healthy individuals, their presence in high levels is associated with various thrombotic, inflammatory and metabolic disorders and with cancer. Besides cancer, these diseases are numerous and range from diabetes and its vascular complications to inflammatory diseases, including atherosclerosis. It concerns, in particular, cardiovascular disease with increased risk of thrombosis or even neurovascular ischemic conditions.
Cellular microparticles may thus be considered as early markers that allow for diagnosing and assessing the risk of developing various diseases, in particular thrombotic, inflammatory and/or metabolic disorders, and thereby enable the therapeutic follow up and monitoring thereof.
Neurovascular injuries and occlusive cardiovascular diseases constitute, right after cancer, the leading causes of morbidity and mortality. In Europe and the United States, these pathologies result in significant costs in the social and economic contexts as well as in terms of public health.
These occlusive accidents are the result of a thrombus formed in situ or embolism. The formation of an occlusive thrombus was associated with localised fibrinolysis defects. However, despite the pathophysiological context, there are no reliable methodologies currently available to assess the fibrinolytic activity in the intravascular space that would enable risk assessment and prevention.
Several studies have shown that high concentrations of cellular microparticles possess good prognostic value.
Methods of detection have been established. Among the few available techniques include flow cytometry, which is the most widely used. This method partially allows phenotyping and partial quantification of cellular microparticles by means of using calibration beads. This method takes advantage of the affinity of Phophatidylserine with annexin A5, a cellular protein. More generally, antibodies directed against antigenic determinants exposed on their membranes are used.
Cellular microparticles can in particular be detected by measuring the fluorescence emitted by annexin A5 or with antibodies labelled with a fluorescent compound.
However, the sensitivity of this method does not allow for the identification of cellular microparticles having a size less than 500 nm.
Annexin A5 is also used in a “capture/quantification” test wherein the cellular microparticles which expose phosphatidylserine are captured by means of annexin A5 immobilised on the surface of microtiter plate wells (test developed by the company Hyphen Biomed).
However, this technique does not allow for the phenotype and distribution of elements to be obtained. Furthermore, its implementation is cumbersome and depends on a number of experimental factors. The capture of cellular microparticles by annexin A5 is highly dependent on calcium ions and is very sensitive to experimental variations (proteolysis, interactions with various ligands, etc.).
Thus, the detection techniques available are lacking in accuracy and sensitivity. Moreover, they are often very expensive.
It is therefore desirable to be able to detect, quantify and characterise, in particular phenotype, in an accurate and standardised manner, cellular microparticles in biological fluids.
The term “biological fluid” is understood to mean any extractable body fluid, including for example, blood, blood plasma, cerebrospinal fluid, bronchoalveolar fluid, urine, synovial fluid, breast milk, saliva, tears, seminal fluid, ascitic fluids, amniotic fluid and effusions (pleural or other). The inventors have now developed a method for detection and/or characterisation in which cellular microparticles are captured by a synthetic ligand.